DE69116187T2 - Use of a dihydropyridine to improve blood circulation in the inner ear - Google Patents

Abstract

Use of a dihydropyridine compound of the general formula: <CHEM> wherein R<1> means a nitrophenyl group; and R<2>, R<3> and R<4> each means a lower alkyl group, or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating or preventing disorder of inner ear microcirculation.

Description

This invention relates to the new use of a dihydropyridine compound for improving the microcirculation of the inner ear.

In particular, this invention relates to a novel use of a dihydropyridine compound (I) represented by the following general formula or a pharmaceutically acceptable salt thereof for improving the microcirculation of the inner ear.

wherein R¹ represents a nitrophenyl group; and R², R³ and R⁴ each represent a C₁-C₆ alkyl group.

The dihydropyridine compound (I) used in this invention is known and described, inter alia, in British Patent No. 2,036,722. It is further known that pharmacologically this dihydropyridine compound has a vasodilatory effect based on calcium ion antagonism and as such is of value as an antianginal or antihypertensive agent, as a cerebral circulation improving agent and further as an antiarteriosclerotic agent.

Intensive researches undertaken by the inventors of the present invention revealed that this dihydropyridine compound (I) and its pharmaceutically acceptable salts have, in addition to the aforementioned effects, the effect of improving the microcirculation of the inner ear. This finding provided the basis for this invention.

The improving effect of the microcirculation of the inner ear of the dihydropyridine compound (I) and its pharmaceutically acceptable salts should be considered as a new pharmacological effect based on the inhibition of the formation of microthrombi, which is considered to be the main factor causing disorders of the microcirculation of the inner ear, and on the prevention of vasospasms. This effect should therefore be considered as pharmacologically different from the effects mentioned above.

It is therefore the object of the present invention to provide a new use of the dihydropyridine compound (I) or a pharmaceutically acceptable salt thereof for the production of a drug for improving the microcirculation of the inner ear. Among these diseases caused by disturbances of the microcirculation of the inner ear, for example, deafness, dizziness and the like are to be mentioned.

The pharmaceutically acceptable salts mentioned above include non-toxic salts of known types, e.g. salts with inorganic bases such as alkali salts (e.g. sodium, potassium, etc.), alkaline earth metals (e.g. calcium, magnesium, etc.), ammonium, etc., and salts with organic bases such as organic amines (e.g. triethylamine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc.).

With respect to the general formula (I) shown above, preferred examples of R¹, R², R³ and R⁴ are as follows.

As examples in which R¹ represents nitrophenyl, 2-nitrophenyl, 3-nitrophenyl and 4-nitrophenyl may be mentioned, and among these, 3-nitrophenyl is particularly preferred.

As examples, where R², R³ and R⁴ are independently As lower alkyl groups, there may be mentioned C₁-C₆ alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, isopentyl, neopentyl, 1- or 2-methylbutyl and hexyl. Preferred are C₁-C₄ alkyl groups. The most preferred example of R² is isopropyl, while methyl is the most preferred example of R³ and R⁴.

The inner ear microcirculation improving agent of the invention can be administered orally or otherwise to humans or other mammals in any conventional pharmaceutical dosage form such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, ointments, suppositories, injections, syrups, etc.

The inner ear microcirculation enhancing agent can be prepared by established pharmaceutical processes using common organic or inorganic carriers well known in the art. These carriers include, among others, various excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc.; binders such as cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc.; disintegrating agents such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, etc.; lubricants such as magnesium stearate, aerosil, talc, sodium lauryl sulfate, etc.; flavorings such as citric acid, menthol, glycine, orange powder, etc.; Preservatives such as sodium benzoate, sodium bisulfite, methylparaben, propylparaben, etc.; stabilizing agents such as citric acid, sodium citrate, acetic acid, etc.; suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc.; dispersing agents such as hydroxypropylmethylcellulose, etc.; diluents such as water etc.; and base waxes such as cocoa butter, white petrolatum, polyethylene glycol, etc.

The dosage of the active ingredient of the dihydropyridine compound (I) depends on such factors as body weight, age of the patient, severity of the disease and route of administration. In general, the microcirculation Inner ear improving agents of the present invention are administered orally in a daily dose of 0.5 to 1000 mg, preferably 1 to 500 mg, as the dihydropyridine compound (I). The single dose in the range of 0.01 to 20 mg, preferably 0.05 to 2 mg per kg body weight is selected.

The following pharmacological test data demonstrate the usefulness of the dihydropyridine compound (I) or a pharmaceutically acceptable salt thereof when used as the inner ear microcirculation improving agent of this invention.

Testing 1 Proceedings

Male Wistar rats (body weights 240-260 g) were anesthetized with pentobarbital. After tracheostomy, the animals were artificially ventilated with a Harvard 683 ventilator. A solution of rose bengal (RB) in saline (10 mg/ml) was infused into the femoral vein at a rate of 24 µmol/kg per hour. After initiation of rose bengal infusion, the left middle ear was opened and the round window lead electrode was fixed in position for electrocochleography. Then, after incising the skin and muscle of the neck region, the tympanic cavity was drilled with a miniature drill and a silver ball electrode was attached to the round glass membrane for use as an active electrode. An indifferent electrode was placed on the center of the back of the head. Thirty minutes after initiation of the Rose Bengal infusion, the lateral wall of the cochlea was irradiated with green light (540 nm) from a light source (L-3306-01A, Hamamatsu Photonics) through an optical fiber. Using a manipulator, the free end of the optical fiber was fixed approximately 5 mm from the lateral wall of the cochlea. In this configuration, the compound action potential (CAP) was recorded at 1 minute intervals. The electrocochleogram was recorded with a Nihon Kohden Neuopack II, averaging 128 clicks of 8 kHz at a sound pressure level of 100 dB. The vehicle (polyethylene glycol (400)-water, 1:1 v/v) or the test compound (dissolved in the vehicle) was administered intravenously 10 minutes before photoexposure. tion injected.

Test connection

6-Cyano-5-methoxycarbonyl-2-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid isopropyl ester (hereinafter referred to as dihydropyridine compound A). Result Dosage (mg/kg) Number of animals Potential for efficacy Time to disappearance (min.) (vehicle only)

Histologically, damage to the stria vascularis of the lateral wall was found after one hour of photoexposure, and complete disintegration of the inner hair cells was observed after 24 hours. These findings represented characteristic ischemic changes of the inner ear, similar to those reported by Robert Kimura (Annalen der otologie, Rhinologie und Laryngologie 67, 6-24, 1958). The ischemic changes were verified morphologically. The fact that pretreatment with heparin (5 minutes before photoexposure) prolonged the time required to attenuate WP suggested that the attenuation of WP was caused by deterioration of the inner ear microcirculation due to a microthrombus formed as a result of injury to the vascular endothelial cells.

Testing 2 Proceedings

Rats were anesthetized with pentobarbital and, while maintaining body temperature at 37ºC with a heating pad, an aqueous solution of rose bengal was continuously infused into the femoral vein. The inner ear was then opened and the tympanic membrane, incus and malleus were without damaging the inner ear.

Using a Hamatsu photonic xenon lamp, the oval window was irradiated with green light of 540 nm via an optical fiber. Using a manipulator, the end of the fiber was fixed approximately 3 mm from the lateral wall of the cochlea.

Photoexposure was started 20 minutes after the start of rose bengal infusion, and a solution of the test compound in polyethylene glycol (400)-water (1:1, v/v) was administered intravenously at the start of photoexposure. Forty minutes after irradiation, both photoexposure and rose bengal infusion were stopped, the wound was sutured, and the animal was allowed to regain consciousness. 24 hours after the cessation of photoexposure, a nystagmus test and a swimming test for imbalance were performed.

Test connection

Dihydropyridine compound A result Dose (mg/kg) Number of test animals Incidence (%) Nystagmus imbalance

From the above results, it is evident that the dihydropyridine compound A improves inner ear microcirculation disorders to prolong the time of disappearance of the active potential (WP) and inhibits the onset of nystagmus and imbalance, and therefore is of value as an inner ear microcirculation improving agent.

The following examples further illustrate the present invention.

example 1

Dihydropyridine compound A 100 g

Hydroxypropylmethylcellulose 500 g

In absolute ethanol (5 liters), the dihydropyridine compound A was dissolved, followed by the addition of hydroxypropylmethylcellulose to provide a suspension. The organic solvent was then distilled off under reduced pressure to give a solid dispersion.

Example 2

Dihydropyridine compound A 100 g

Hydroxypropylmethylcellulose 500 g

Sucrose 9.4kg

In absolute ethanol (5 liters), the dihydropyridine compound A and hydroxypropylmethylcellulose were suspended, followed by the addition of sucrose, and the mixture was stirred. The organic solvent was then distilled off under reduced pressure to provide a solid dispersion. This dispersion was processed into fine granules by established pharmaceutical procedures.

Example 3

Dihydropyridine compound A 100 g

Hydroxypropylmethylcellulose 500 g

Lactose 6.87kg

low substituted hydroxypropyl cellulose 1.5 kg

Magnesium stearate 30 g

In absolute ethanol (5 liters) were suspended the dihydropyridine compound A and hydroxypropylmethylcellulose, followed by the addition of lactose and low substituted hydroxypropylcellulose, and the resulting mixture was stirred. The organic solvent was then distilled off under reduced pressure to give a solid dispersion. This dispersion was granulated by a routine procedure and after the addition of magnesium stearate, the granulation was routinely compressed into tablets. Each of these tablets contained 2 mg of the dihydropyridine A.

Example 4

The tablets prepared in Example 3 were each film coated by a routine process using a coating composition comprising hydroxypropylmethylcellulose (5.1 mg), titanium dioxide (1.6 mg), polyethylene glycol 6000 (0.8 mg), talc (0.4 mg) and yellow iron oxide (0.1 mg) to provide film-coated tablets each containing 2 mg of dihydropyridine Compound A.

Example

The racemate of 5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester (16.7 g) and cinchonidine (14.4 g) was dissolved in methanol (100 mL) and the solution was refluxed for 15 minutes. The reaction mixture was then left to stand at room temperature. The resulting precipitate was collected by filtration, washed with methanol and air dried to give (-)-5- carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester cinchonidine salt (11.74 g).

The mother liquor was distilled under reduced pressure to remove the solvent. The crystalline residue was washed with a mixture of ethyl acetate and diisopropyl ether, diluted with 2N hydrochloric acid (40 ml) and extracted with ethyl acetate. The extract was washed with aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and distilled under reduced pressure to remove the solvent. The procedure gave a mixture of (+) and (-) isomers of methyl 5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylate (11.15 g).

This mixture (11.15 g) and cinchonine (9.54 g) were dissolved in ethyl acetate with heating, and the solution was left at room temperature. The resulting precipitate was collected by filtration, washed with ethyl acetate and recrystallized from ethanol. The procedure afforded (+)-5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester cinchonine salt (7.62 g).

M.p.: > 164ºC (decomposition)

[α]D²&sup0; : +243.2º (c=1.0, CH₃OH)

Example 6

In ethyl acetate (50 ml) was suspended (+)-5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester cinchonine salt (7.41 g) with stirring, followed by the addition of 2N hydrochloric acid (20 ml) with stirring, and the water layer was then removed. The organic layer was washed with aqueous sodium chloride solution, dried over magnesium sulfate and distilled under reduced pressure to remove the solvent. The procedure gave (+)-5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester (3.67 g).

M.p.: 203 ºC (decomposition)

[α]D²&sup0; : +234.1º (c=1.0, CH₃OH)

NMR (DMSO-d6, δ): 2.34 (3H, S), 3.71 (3H, S), 5.13 (1H, s), 7.56 - 7.82 (2H, m), 7.91 - 8.25 (2H, m), 10.25 (1H, broad s)

Example 7

In methylene chloride (30 ml) was suspended (+)-5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester (3.12 g), followed by the addition of phosphorus pentachloride (2.46 g) under ice-cooling, and the mixture was stirred for 30 minutes. Then, a solution of isopropyl alcohol (1.4 g) in methylene chloride (10 ml) was added dropwise over a period of 10 minutes. The mixture was stirred for 20 minutes. After this time, a 5% aqueous sodium carbonate solution (30 ml) was added, and the mixture was stirred at room temperature for 1 hour. The organic layer was separated, and the aqueous layer was extracted with methylene chloride. The organic layers were combined, washed with aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to remove the solvent. The residue was purified by silica gel (75 g) column chromatography, eluting with benzene-ethyl acetate (10:1). The solvent was removed by the eluate was removed under reduced pressure and the residue was crystallized from diisopropyl ether to give (+)-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester-5-carboxylic acid isopropyl ester (3.34 g).

Melting point: 120-122ºC

[α]D²&sup0; : +222.4º (c=1.0, CH₃OH)

NMR (CDCl₃, δ): 1.09 (3H, d, J=6.5 Hz), 1.26 (3H, d, J=6.5 Hz), 2.40 (3H, s), 3.76 (3H, s), 4.97 (1H, heptet, J=6.5 Hz), 5.17 (1H, s), 6.96 (1H, broad s), 7.21 - 7.77 (2H, m), 7.95 - 8.21 (2H, m)

Elemental analysis: for C₁�9H₁�9N₃O�6

Calculated:C 59.22; H 4.97; N 10.90;

found: C 59.38; H 5.08; N 10.98;

Example 8

The (-)-5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester cinchonidine salt (11.74 g) prepared according to Example 5 was recrystallized from methanol to give the pure compound (9.36 g).

Melting point: 159-160ºC

[α]D²&sup0; : -198.9º (c=1.0, CH₃OH)

This compound (9.05 g) was suspended in ethyl acetate (50 mL) followed by the addition of 2N hydrochloric acid (20 mL). The aqueous layer was discarded. The organic layer was washed with aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The procedure gave (-)-5-carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester (5.11 g).

M.p.: 205ºC (decomposition)

[α]D²&sup0; : -230.7º (c=1.0, CH₃OH)

NMR (DMSC-d6, δ): 2.34 (3H, s), 3.71 (3H, s), 5.13 (1H, s), 7.56 - 7.82 (2H, m), 7.91 - 8.23 (2H, m), 10.25 (1H, broad 5)

Example 9

(-)-5-Carboxy-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid methyl ester (4.47 g) was reacted with phosphorus pentachloride (3.62 g) and isopropyl alcohol (2.5 g) in the same manner as in Example 7 to give (-)-2-cyano-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-5-carboxylic acid isopropyl ester-3-carboxylic acid methyl ester (4.9 g).

Melting point: 120-122ºC

[α]D²&sup0; : -219.6º (c=1.0, CH₃OH)

NMR (CDCl₃, δ): 1.09 (3H, d, J=6.5 Hz), 1.25 (3H, d, J=6.5 Hz), 2.39 (3H, s), 3.78 (3H, s), 4.98 (1H, heptet, J=6.5 Hz), 5.19 (1H, s), 7.0 (1H, broad s), 7.25 - 7.76 (2H, m), 7.96 - 8.21 (2H, m)

Elemental analysis for C₁�9H₁�9N₃O�6

calculated: C 59.22; H 4.97; N 10.90;

found: C 59.17; H 4.92; N 10.91;

Claims (3)

1. Use of a dihydropyridine compound of the general formula: wherein R¹ represents a nitrophenyl group; and R², R³ and R⁴ each represent a C₁-C₆ alkyl group, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of disorders of the microcirculation of the inner ear. 2. Use according to claim 1, wherein the disturbance of the microcirculation of the inner ear is hearing loss or dizziness. 3. Use according to claim 1 or 2, wherein the dihydropyridine compound (I) is 6-cyano-5-methoxycarbonyl-2-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid isopropyl ester.